The gut microbiota (GM) plays a pivotal role in modulating both the efficacy and toxicity of chemotherapy (CT) in pediatric oncology patients, underscoring the relevance of this review. The authors analyzed and systematized current literature data on the role of GM in treatment tolerance and efficacy, particularly in acute leukemia (AL). The existence of a complex bidirectional relationship between CT and GM is demonstrated. CT induces dysbiotic shifts in the GM, characterized by reduced alpha-diversity, depletion of commensal genera, and an increase in the abundance of opportunistic pathogens, which serves as a predictor of complications such as mucositis, febrile neutropenia, and sepsis. Conversely, GM exerts influence on CT through direct effects on the metabolism of cytostatic agents and systemic immunomodulation. A key mechanism of GM's influence is the production of short-chain fatty acids (SCFAs), particularly butyrate, which enhances the function of cytotoxic T-lymphocytes, induces apoptosis in tumor cells, and reinforces intestinal barrier integrity. Promising strategies for correcting dysbiosis include probiotics, prebiotics, and fecal microbiota transplantation (FMT), the latter demonstrating high efficacy in steroid-refractory graft-versus-host disease (GVHD). The integration of microbiome-based biomarkers into predictive models and the application of multi-omics technologies pave the way for the development of personalized treatment approaches. In conclusion, GM is a significant factor determining the outcomes of CT in children, and strategies aimed at its modulation hold considerable potential for improving patient survival and quality of life, although further validation through randomized controlled trials is warranted.